Discovery of Benfotiamine as a subnanomolar P2Y14R antagonist for inflammatory diseases via drug repurposing and molecular dynamics-guided mechanism elucidation.

Integrated computational-experimental platforms are promising for GPCR drug discovery, but systematically connecting high-resolution structural prediction with functional validation remains challenging. Here, we apply an integrated platform to discover a novel, potent antagonist targeting the P2Y14 receptor (P2Y14R), a key target in inflammatory diseases. Combining structure-based virtual screening, BPMD, all-atom molecular dynamics simulations, and cellular cAMP functional assays, our platform pinpointed Benfotiamine, an approved drug, as a nanomolar-potency P2Y14R antagonist (IC50 = 0.31 nM). Computational analyses indicated that Benfotiamine exerts a "conformational lock" to stabilize the inactive receptor and hinder Gi coupling. Subsequent CETSA and SPR experiments confirmed its direct binding to the target. Functionally, Benfotiamine exhibited therapeutic efficacy in murine models of dextran sulfate sodium-induced colitis and monosodium urate-induced gouty arthritis. Beyond presenting a promising repurposed anti-inflammatory agent, this study validates a modular and extensible computational-experimental integration platform that can be widely applied to expedite the discovery and mechanistic characterization of GPCR-targeted therapeutics.